So-called biosensors utilize molecular recognition abilities possessed by biological substances. Such biologic substances include nucleic acid molecules including genes (e.g., DNA and RNA), aptamers, sugar-binding proteins, enzymes, and antibodies. Many researches and developments have been conducted on the biosensors with the aim of extensive application thereof.
With the increased interest in environmental pollutant problems, social safety, and health, further technical developments have been demanded increasingly for biosensors. The goal thereof is, for example, to apply these biosensors to diverse targets to be detected.
Specifically, detection apparatuses have been developed widely using the selectivity of molecular recognition possessed by each biological substance molecule, as described above. Examples thereof include DNA sensor chips. These DNA sensor chips utilize the nucleotide sequence-dependent complementary hydrogen bond between deoxyribonucleic acid (hereinafter, referred to as DNA) chains (hybridization reaction between complementary chains) Alternative examples thereof include antibody sensors or protein chips. These apparatuses utilize molecular recognition abilities derived from the specific binding abilities between protein molecules and low-molecular-weight compounds or between protein molecules. For example, the apparatuses utilize antigen-antibody reaction or sugar-lectin binding to detect disease markers or the like eluted into blood. Further examples thereof include the developments of detection apparatuses based on a variety of detection approaches, including biosensors such as enzyme sensors. The enzyme sensors utilize oxidoreductase or hydrolase to detect substrate substance concentrations. These biosensors are typified by glucose sensors for diabetes mellitus patients.
Current biosensors utilizing these biological substances generally adopt a system using the form of a biological substance-immobilized base substance. In this system, the biological substances used (e.g., nucleic acid molecules such as DNA or proteins such as antibodies or enzymes) are immobilized on the surface of a base substance such as a substrate or carrier.
Alternatively, one example of performance qualities required for biosensors currently under development includes high sensitivity and size reduction. The important technical challenge to this goal of “high sensitivity and size reduction” is to effectively use the space of a very small reaction field or detection field and to enhance detection sensitivity. The aim of size reduction is to reduce the amounts of samples collected from patients so as to ease the burden on the patients. As a result, techniques have been demanded which are capable of detecting, with high sensitivity, target molecules present in very small amounts of samples.
Examples of methods for immobilizing biological substances, particularly, proteins, on a substrate include an approach which involves: forming a coating layer of a protein solution on substrate surface; then removing the solvent contained in the coating layer; and drying the substrate so as to immobilize the proteins on the substrate surface through physical adsorption. Alternative examples thereof include an approach which involves: for the purpose of introducing reactive functional groups, chemically modifying substrate surface or protein molecules; and then forming a chemical bond using the reaction between the introduced reactive functional groups so as to immobilize the protein molecules on the substrate surface through chemical bonds. An alternative previously known approach is the immobilization of proteins onto substrate surface using molecular recognition.
Patent Document 1 discloses one example of an immobilization method based on physical adsorption. This document discusses a method for producing an enzyme electrode. This method utilizes an approach which involves immobilizing enzyme proteins through physical adsorption on the surface of a conductive substrate via an organic charge-transfer complex layer formed on the conductive substrate surface.
Non-Patent Document 1 discloses one example of an immobilization method using a chemical bond. This document discusses an immobilization method which involves forming, using a cross-linking agent, the chemical bond between an amino group derived from an amine-based silane coupling agent provided on the platinum-deposited surface of a silicon substrate and a peptide chain. In addition, detection apparatuses such as biosensors obtained by immobilizing antibodies onto a glass substrate may be prepared using such an immobilization method. In this case, reactive functional groups are introduced into the surface of a glass substrate by treatment with a silane coupling agent. Then, peptide chains are immobilized thereon via a chemical bond using a cross-linking agent.
Patent Document 2 discloses one example of an immobilization method using molecular recognition. This document discusses a technique of specifically immobilizing proteins with orientation onto a substrate through molecular recognition abilities imparted by fusing the proteins with affinity peptides having affinity for silicon oxide arranged on the substrate surface. This method can maintain the maximum target substance-binding abilities of the proteins.
While immobilization techniques have made progress, detection techniques for increasing the added value of biosensors have been developed acceleratingly. For example, the non-labeling detection of target substances provides reduction in the number of detection steps, reduction in time, and kinetic analysis. For the purpose of further achieving detection with high sensitivity or low-molecular-weight compound detection, the development of biosensors has been attempted using a localized surface plasmon resonance (hereinafter, referred to as LSPR) phenomenon.
Non-Patent Document 3 discloses the detection of biotin-avidin biological reaction using an LSPR element. In this LSPR element, nano-order metal structures (rings and dots) are arranged on substrate surface. Alternatively, Non-Patent Document 4 has attempted the development of more effective sensors. In this document, LSPR elements having various shapes are evaluated for electric field strength distribution by calculation. Alternatively, techniques of applying Hall elements to biosensors have also been known. The Hall elements, one of semiconductor magnetic sensors, utilize the Hall effects. The aim of the techniques is to basically quantitatively detect, with ultra-high sensitivity, target substances labeled with magnetic substances (Non-Patent Document 2 and Patent Document 3). Moreover, Non-Patent Document 5 discusses more effective sensors. In this document, positions within magnetically sensitive surface, Hall voltages, and magnetic flux density distribution are evaluated by calculation.
Alternatively, Patent Document 4 discloses one example of a reaction promotion method. This document discusses a technique of detecting biomolecular interaction in a short time in real time. In this technique, the dielectrophoresis of biomolecules is induced by applying a high-frequency alternating voltage thereto. As a result, very small amounts of target substances are concentrated so as to promote the reaction.
However, such a detection technique or reaction promotion method basically has inhomogeneous distribution, such as a gradient of electric field or magnetic field strength, in a sensing or reaction promotion region. Therefore, the conventional techniques of immobilizing biological substances onto these regions are not necessarily optimized in terms of quantitative detection or the like. The important challenge to the increase of the added value of biosensors can be to develop more effective techniques of immobilizing biological substances. Specifically, protein immobilization techniques have been demanded which are more precise or effective, particularly, as the whole biosensor involving a detection unit or reaction promotion unit, in addition to orientational or homogeneous immobilization techniques, such as the conventional immobilization techniques.    Patent Document 1: Japanese Patent Application Laid-Open No. 06-003317    Patent Document 2: Japanese Patent Application Laid-Open No. 2005-95154    Patent Document 3: Japanese Patent Application Laid-Open No. 2006-234762    Patent Document 4: Japanese Patent Application Laid-Open No. 2006-145400    Non-Patent Document 1: Sensors and Actuators B 15-16 p127 (1993)    Non-Patent Document 2: IEEE TRANSACTIONS ON MAGNETICS, VOL. 41, NO. 10, October 2005    Non-Patent Document 3: Langmuir 2006, 22, 7109-7112    Non-Patent Document 4: Journal of Fluorescence Vol. 14 No. 4 (2004)    Non-Patent Document 5: Journal of Applied Physics Vol. 83 No. 11 (1998) p 6161-p 6165